Detection and identification of microbes in prosthetic joint infections by culture and molecular methods

Bacterial biofilms have been observed in many device-related infections including orthopedic implants. This mode of growth makes the infection difficult to treat and constitutes a challenge to current sampling procedures and culture practices to obtain a reliable diagnosis. The aim of the study was to use molecular methods and standard microbiological culture to investigate the microbial diversity in surgical samples (eg. synovial fluid, periprosthetic tissue, removed prosthesis) collected from different revision arthroplasties. A total of 75 specimens from 37 surgical procedures were analyzed by broad range 16S rRNA gene PCR, which detected bacteria in 29 specimens from 17 procedures. Subsequent cloning and Sanger sequencing identified ~50 bacterial species. Concomitant samples from 14 of the 37 procedures yielded positive culture and identified 7 species in total. Samples from additional 10 surgical procedures were examined using IBIS universal biosensor, 454 pyrosequencing of 16S rRNA gene and culture. IBIS, pyrosequencing and culture detected the presence of bacteria in samples from 8, 2 and 2 procedures, and identified 11, 2 and 3 species, respectively. All in all, molecular methods uncovered many more species including known pathogens and species not previously reported in orthopedic infections, and polymicrobial communities were commonly observed. Additionally the molecular findings suggested the bacterial composition and yield varied depending on the position and type of samples used for analysis, particularly prosthetic implants yielded more positive results. Furthermore, the presence of bacteria as both single cells and microcolonies were visualized by fluorescence in situ hybridization using peptide nucleic acid and DNA probes and confocal scanning laser microscopy, indicating biofilm formation. In conclusion, this study indicated that to improve the microbiological diagnosis of prosthetic joint infections molecular methods may be useful supplements to routine cultures, and the current intraoperative sampling strategy needs to be optimized.